Optimisation of growing conditions for the apple rootstock M26 grown in RITA containers using temporary immersion principle

The use of bioreactors may provide an efficient and economic tool for mass clonal propagation of plants if technical problems can be solved. In this paper, we report the results of experiments aimed at optimising conditions for apple rootstock M26 grown in RITA containers using the temporary immersion principle. We tested different types and sizes of explants, different concentrations of plant growth regulators (BAP, kinetin and IBA) in the multiplication and elongation phases, and medium exchange during the shoot elongation period. The results show that the higher concentrations of cytokinins were required during the shoot multiplication phase, while the lower concentrations were better during the shoot elongation phase. Hyperhydricity was increased with increasing concentration in of cytokinins during both shoot multiplication and shoot elongation phases. The best shoot production in terms of shoot number and shoot quality was obtained using 4.4mol BAP and 0.5mol IBA during the shoot multiplication phase and 1.1mol BAP and 0.25mol IBA during the shoot elongation phase. Medium exchange twice during the shoot elongation phase resulted in higher shoot production compared with no exchange of the medium. However, it also resulted in increased hyperhydricity. Immersion frequency of 16 times per day gave a higher multiplication rate and longer shoots than 8 times per day. The explant size of 0.5cm or 1cm resulted in a significantly higher shoot production rate compared with that of 1.5cm, but shoot length and hyperhydricity were not affected by the explant size. Shoot cultures from the liquid media rooted normally in the RITA containers with more than 90% rooting and the rooted plantlets acclimatised well in the greenhouse.     

Physiology of effects of temporary immersion bioreactors on micropropagated pineapple plantlets

Summary Temporary immersion bioreactors are an efficient tool for plant mass propagation because they increase multiplication rate and plant quality. Little knowledge is available on the ecosystem and physiological behavior of plantlets when using this new culture technique. In order to evaluate the effects of the conditions on physiological change of pineapple plantlets, a factorial experiment was conducted, where axillary clusters were cultured under two levels of photosynthetic photon flux (PPF): 30 μmol m⁻²s⁻¹ (low) and 225 μmol m⁻²s⁻¹ (high), using two culture methods (conventional micropropagation in liquid medium and a temporary immersion bioreactor) during the elongation phase. CO₂ concentration in the headspace volume container was measured during a whole cycle of temporary immersion (3h). At the time before the next immersion period, the levels of CO₂ increased significantly to 14171 μmol mol⁻¹ at high PPF. The maximal photosynthetic rate as well as the maximum quantum yield of photosystem II were low for plantlets cultivated in the femporary immersion bioreactor at high PPF. However, these plantlets showed large increases in sugar and nitrogen uptake and also increases in dry weight and foliar area. These results indicate that shoot growth did not totally depend on the photosynthesis process. In vitro pineapple plantlets appeared to use more nutrients in the culture medium than those from photosynthesis. In summary, temporary immersion bioreactor-derived plantlets showed remarkable nutrient uptake, indicating a higher photo-mixotrophic metabolism.     

Exploring natural variation of Pinus pinaster Aiton using metabolomics: Is it possible to identify the region of origin of a pine from its metabolites?

Natural variation of the metabolome of Pinus pinaster was studied to improve understanding of its role in the adaptation process and phenotypic diversity. The metabolomes of needles and the apical and basal section of buds were analysed in ten provenances of P. pinaster, selected from France, Spain and Morocco, grown in a common garden for 5 years. The employment of complementary mass spectrometry techniques (GC‐MS and LC‐Orbitrap‐MS) together with bioinformatics tools allowed the reliable quantification of 2403 molecular masses. The analysis of the metabolome showed that differences were maintained across provenances and that the metabolites characteristic of each organ are mainly related to amino acid metabolism, while provenances were distinguishable essentially through secondary metabolism when organs were analysed independently. Integrative analyses of metabolome, environmental and growth data provided a comprehensive picture of adaptation plasticity in conifers. These analyses defined two major groups of plants, distinguished by secondary metabolism: that is, either Atlantic or Mediterranean provenance. Needles were the most sensitive organ, where strong correlations were found between flavonoids and the water regime of the geographic origin of the provenance. The data obtained point to genome specialization aimed at maximizing the drought stress resistance of trees depending on their origin.     

A temporary immersion plant propagation bioreactor with decoupled gas and liquid flows for enhanced control of gas phase

Temporary immersion bioreactors (TIBs) are being used to propagate superior plant species on a commercial scale. We demonstrate a new TIB design, a Hydrostatic‐driven TIB (Hy‐TIB), where periodic raising and lowering the media reservoir maintains the advantages of temporary immersion of plant tissues without requiring large amounts of gas to move the media that is a characteristic of other TIB designs. The advantage of utilizing low volumes of gas mixtures (that are more expensive than air) is shown by a doubling of the growth rate of plant root cultures under elevated (40%) oxygen in air, and with CO₂ supplementation showing improved phototrophic and photomixotrophic growth of seedless watermelon meristem cultures. The development of this bioreactor system involved overcoming contamination issues associated with utilizing very low gas flow rates and included utilizing microchip pressure sensors to diagnose unexpected changes in internal bioreactor pressure (± 20 Pa ～0.0002 atm) caused by flexing of non‐rigid plastic bag vessels. The overall design seeks to achieve versatility, scalability and minimum cost such that bioreactor technology can play an increasing role in the critical need to improve plant productivity in the face of increasing demand for food, reduced resources, and environmental degradation. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:337–345, 2016     

Cycle characteristics in a temporary immersion bioreactor affect regeneration,morphology, water and mineral status of coffee (<Emphasis Type="Italic">Coffea arabica</Emphasis>) somatic embryos

Mass regeneration of Coffea arabica L. somatic embryos using a temporary immersion bioreactor was improved by optimizing the immersion cycles, i.e. both the duration and the frequency of immersions. It was demonstrated that increasing the frequency of short immersions (1 min immersions every 24, 12 and 4 h) stimulated embryo production (480, 2,094 and 3,081 embryos/1-l bioreactor, respectively) and improved quality (60, 79 and 85 of torpedo shaped embryos, respectively). On the other hand, an increase in the immersion duration (1, 5 and 15 min) inhibited embryo regeneration (from 2,094 to 428 embryos per 1-l bioreactor) and negatively affected their morphological quality (from 79 to 49 torpedo-shaped embryos) and the conversion of embryos into plants (from 70 to 33). A 15 min immersion duration applied every 4 h produced hyperhydric symptoms in 90 of the embryos. Hyperhydric embryos were characterized by higher fresh weight and water content, more negative values for water potential and higher K⁺ content when compared to normal torpedo-shaped embryos. Micrographs showed structural problems in the globular stage, such as the existence of an irregular epidermis and an absence of reserves. Whatever the immersion cycle used, the somatic embryos exhibited water and mineral characteristics very different from those of their zygotic counterparts. The use of 1 min immersions every 4 h led to the production of the largest quantities of torpedo-shaped embryos without hyperhydricity that succeeded in regenerating plants (75 conversion).     

LC-MALDI-TOF MS-Based Rapid Identification of Phenolic Acids

This study is the first on combined HPLC and MALDI-TOF MS analysis of phenolic acids. The analyses were carried out for phenolic acid mixtures and showed a unique, individual co-crystalline pattern for each phenolic acid. HPLC could distinguish phenolic acids and MALDI-TOF MS provided comparable mass (m/z) profiles for the samples. This combined study proved to be rapid in the accurate identification and structural analysis of phenolic acids with different masses.     

Flavonoids from the leaves of Cyclanthera pedata: two new malonyl derivatives

Reversed-phase HPLC coupled with electrospray MS has been used for the simultaneous separation and determination of flavonoid metabolites in leaves of Cyclanthera pedata, an edible Peruvian plant mainly used in South America for its anti-inflammatory, hypoglycaemic and hypocholesterolaemic properties. The flavonoid content of the leaves of C. pedata was compared qualitatively and quantitatively with that of the fruits. The isolation and structural characterisation by MS and NMR of two new minor components of the fruits, namely, 6-C-fucopyranosyl-(3-malonyl)-chrysin and 6-C-fucopyranosyl-(4-malonyl)-chrysin, are described.